Proc. Natl. Acad. Sci. USA Vol. 87, pp. 9863-9867, December 1990 Biochemistry Cytochrome a, of Acetobacter aceti is a cytochrome ba functioning as ubiquinol oxidase (bacterial terminal oxidase/puriflcation/proteoliposome/electrochemical proton gradient) KAZUNOBU MATSUSHITA, EMIKO SHINAGAWA, OSAO ADACHI, AND MINORU AMEYAMA Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan Communicated by Britton Chance, September 17, 1990 ABSTRACT Cytochrome a, is a classic cytochrome that in tochrome a, itself (9). Until now, cytochrome a, has not been the 1930s had already been detected in Acetobacter strains and biochemically characterized. in the 1950s was identified as a terminal oxidase. However, Acetic acid bacteria are classified into two genera- recent studies did not substantiate the previous observations. Acetobacter and Gluconobacter; the latter has been shown to We have detected a cytochrome a,-like chromophore in Ace- contain a ubiquinol-oxidizing cytochrome o as the sole ter- tobacter acedi, which was purified and characterized in this minal oxidase (11, 12). Acetobacter has been reported to be study. The cytochrome was solubilized from membranes of the subdivided into two classes: one contains cytochrome d and strain with octyl fi-D-glucopyranoside and was purified by the other contains only an a1-like component (13). Recently, single column chromatography. The purified cytochrome ex- we observed that Acetobacter aceti contains an a1-like cy- hibited a broad a peak around 600-610 nm, which turned to tochrome when the cells are grown with shaking but not when a sharp peak at 589 nm in the presence of cyanide. Carbon they are grown statically (unpublished data). Thus, in this monoxide difference spectra of the cytochrome indicated the study, we purify and characterize the a1-like cytochrome of presence of an a-type cytochrome. The cytochrome contained A. aceti. Our results show that the cytochrome is the so- 1 mol each ofhemes b and a and probably one copper ion. These called cytochrome a,, which is also shown to be a ubiquinol results suggest that the cytochrome purified from A. acedi is the oxidase consisting of hemes b and a. so-called cytochrome a,, and thus the existence of the classic cytochrome has been reconfirmed. The purified enzyme con- MATERIALS AND METHODS sisted of four polypeptides of 55, 35, 22, and 18 kDa, and it showed a sedimentation coefficient of 6.3 in the native form. Materials. Ubiquinone 2 (Q2) was kindly supplied by Eizai S (Tokyo) and the reduced form (Q2H2) was prepared as The enzyme had a high ubiquinol oxidase activity (140-160 described by Rieske (14). Octyl /3-D-glucopyranoside (octyl jimol ofubiquinol-2 oxidized per min per mg ofprotein). When glucoside), valinomycin, and nigericin were purchased from reconstituted into proteoliposomes, the cytochrome could gen- Calbiochem. 3,3'-Diisopropylthiodicarbocyanine [diS-C3(5)] erate an electrochemical proton gradient during oxidation of was from Molecular Probes. Dansylglycine and 2-heptyl-4- ubiquinol. Thus, cytochrome a1 of A. acedi has been shown to hydroxyquinoline N-oxide (HQNO) were from Sigma, 3-un- be a cytochrome ba terminal oxidase capable of generating an decyl-2-hydroxy-1,4-naphthoquinone (UHNQ) was from Al- electrochemical proton gradient concomitant with ubiquinol drich, and DEAE-Toyopearl was from Toyo Soda (Tokyo). oxidation. All other materials were of analytical grade and were ob- tained from commercial sources. Cytochrome a, has a long history, going back to the classic Bacterial Strain, Growth Conditions, and Preparation of work of Warburg showing that Acetobacter pasteurianum Membranes. A. aceti IFO 3284 was grown to the late- (syn. Acetobacter pasteurianus) contains a weak band at 589 logarithmic phase at 30'C in a shaking culture medium nm, which is intensified by cyanide and shifts to 592 nm in the consisting of 1% glycerol/0.3% yeast extract/0.2% polypep- presence of carbon monoxide (for reviews, see refs. 1-3). In tone/0.1% KH2PO4/0.1% K2HPO4. Membranes were pre- the 1950s, the earlier finding was confirmed by Chance (4-6), pared by suspending cells in 50 mM potassium phosphate who also showed that cytochrome a, works as a terminal buffer (pH 6.5) containing 5 mM MgSO4 and some DNase, oxidase in A. pasteurianus. Thereafter, several other bacte- passing them through a French press, and then centrifuging rial strains were reported to contain cytochrome a,, judging at 80,000 x g for 90 min (11). from the a band, which ranges from 585 to 596 nm (for Purification of Cytochrome a,. All steps were performed at reviews, see refs. 1 and 2). Recently, however, a1-like 0-40C. Membranes were homogenized with 10 mM potas- hemoprotein detected in Escherichia coli has been shown to sium phosphate buffer (pH 6.0) at a protein concentration of be ascribable to heme b contained in the cytochrome d 10 mg/ml, and Triton X-100 was added to the suspension at terminal oxidase complex (7), and that of Nitrosomonas a final concentration of 1%. After mixing on ice for 30 min, europaea has been proved to be a cytochrome aa3 (8, 9). the suspension was centrifuged at 80,000 x g for 60 min. The Furthermore, even in A. pasteurianus, a recent study did not resultant precipitate was homogenized with 50 mM potas- prove the presence of cytochrome a, but, instead, showed sium phosphate buffer (pH 6.5) at a protein concentration of that a1-like pigment works to donate electrons to cytochrome 10 mg/ml, and octyl glucoside was added at a final concen- d terminal oxidase (10). Thus, an increasing body ofevidence tration of 1.25%. The suspension was mixed on ice for 30 min has shown that almost all of these a1-like hemoproteins are and then centrifuged as described above. The resultant not cytochrome a, (for review, see ref. 1), and furthermore supernatant was directly applied onto a DEAE-Toyopearl there is a report that casts doubt on the presence of cy- Abbreviations: Q, or QH2, ubiquinone n or ubiquinol n; octyl glucoside, octyl/3-D-glucopyranoside; diS-C3-(5), 3,3'-diisopropylthi- The publication costs of this article were defrayed in part by page charge odicarbocyanine; HQNO, 2-heptyl-4-hydroxyquinoline N-oxide; payment. This article must therefore be hereby marked "advertisement" UHNQ, 3-undecyl-2-hydroxy-1,4-naphthoquinone; TMPD, N,N,N', in accordance with 18 U.S.C. §1734 solely to indicate this fact. N'-tetramethyl-p-phenylenediamine. 9863 Downloaded by guest on September 23, 2021 9864 Biochemistry: Matsushita et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 1. Purification of cytochrome a1 from the membranes of A. aceti P. Protein, Q2H2 oxidase activity 'Fx000S.f- ~ - I Step mg Units Units/mg IM:i .IlJ, Membrane 118 519 4.4 Washed membrane 89.3 474 5.3 Octyl glucoside extract 18.6 444 23.9 4fte -14} 11 DEAE-Toyopearl 2.8 403 144.0 Enzyme activity is expressed as the activity unit to oxidize 1 ;Lmol -1I/ of quinol per min at 25TC. column (-1-ml bed vol per 2 mg of protein) that had been -.... .. .. -I V equilibrated with 50 mM potassium phosphate buffer (pH 6.5). Then, the column was washed with 150 mM potassium phosphate buffer (pH 6.5) containing 1% octyl glucoside at a flow rate of -20 ml/hr. The enzyme having Q2H2 oxidase FIG. 1. SDS/PAGE of cytochrome a1 purified from A. aceti. activity was eluted at a 3-4 column vol after the elution of Right lane, purified oxidase (8 ,ug of protein) was stained with nonadsorbed impurities. Fractions having a specific activity Coomassie blue. Left lane, Coomassie blue-stained marker proteins >140 units/mg were pooled, concentrated by ultrafiltration (2 ,ug of protein each) of phosphorylase b, bovine serum albumin, (Toyo UK 50 membrane filter), and stored in liquid nitrogen. ovalbumin, carbonic anhydrase, and lysozyme. Reconstitution of Cytochrome a, into Proteoliposomes. Cy- tion analysis. Protein content was determined by a modified tochrome a1 (52 ng) purified as described above was mixed Lowry method (16). with sonicated E. coli phospholipids (5 mg), and octyl glu- coside was added to a final concentration of 1.25% in a total vol of 2 ml. The mixture was incubated for 30 min on ice and RESULTS then dialyzed overnight at 4TC against 500 ml of 50 mM Purification and Molecular Properties of Cytochrome a,. potassium phosphate buffer (pH 6.5). The dialysate was Cytochrome a1 was solubilized by octyl glucoside from the centrifuged at 120,000 x g for 3 hr, and the precipitate was membranes prewashed with Triton X-100 and then purified suspended with the same buffer in a total vol of 0.4 ml. The by DEAE-Toyopearl column chromatography. This purifi- suspension was rapidly frozen in liquid nitrogen, thawed, and cation procedure is essentially the same as one used for the briefly sonicated immediately before use. Analytical Procedures. Oxidase activity for Q2H2 and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was 428 measured spectrophotometrically at 250C by following the 422 increase in absorbance at 275 and 520 nm, respectively. The reaction mixture (total, 1 ml) contained 50 mM potassium phosphate buffer (pH 6.5), 30 /iM quinol or 3.6 mM TMPD, 1:429 0.02% Tween 20, and the enzyme. Activity was calculated by using a millimolar extinction coefficient of 12.25 for quinol or 6.1 for TMPD. Membrane potential (inside negative) and pH 414 gradient (inside alkaline) were measured by following the fluorescence quenching of diS-C3-(5) and the fluorescence intensity of dansylglycine as described (12). Absorption IWl 591 spectrophotometry was performed with a Hitachi 557 dual- wavelength spectrophotometer. Low-temperature difference Z spectra were recorded in liquid nitrogen with the same 400 0.05k 560 612 photometer using a cuvette with a 2-mm light path.